Two multienzyme systems for DNA replication based on purified Xenopus laevis DNA replication enzymes will be reconstituted in vitro. The first of these, based on single-stranded circular DNA templates, will be used to investigate priming, both of Okazaki fragments and of replicons at DNA origins. The second, based on double stranded circular DNA templates, will be used to investigate the role of nicking-closing enzyme, helix destabilizing enzyme, and other proteins affecting torsional constraints during DNA replication. Both systems will be based entirely on proteins and DNA molecules isolated from oocytes and eggs of the frog, Xenopus laevis. The proteins involved include DNA polymerases alpha, beta, gamma, and delta, nicking-closing enzyme, ribonuclease H, DNA binding protein, DNA ligase, and DNA primase.